Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 935
Frequency-domain lifetime measurements, 157–204
apparent phase and modulation lifetime, 191–192
background correction, 169–170
biochemical examples, 186–189
DNA, DAPI-labeled, 186–187
fluorescence microscopy with LED light source, 189
Mag-Quin-2, magnesium probe, 187–188
melittin lifetime distributions, cross fitting of models, 188–189
recovery of lifetime distributions from data, 188
color effects, 168–169
gigahertz fluorometry, 175–178
biochemical examples, 177–178
measurements, 177
instrumentation, 163–168
cross-correlation detection, 166
frequency synthesizers, 167
history, 163–164
light modulators, 165–166
200-MHz fluorometers, 164–165
photomultiplier tubes, 167–168
principles of measurement, 168
radio-frequency amplifiers, 167
multi-exponential data analysis, 178–186
global analysis of two-component mixture, 182–183
two closely spaced lifetimes, 180–182
two widely spaced lifetimes, 178–180
multi-exponential decay analysis
maximum entropy analysis, 185–186
three-component mixture, limits of resolution, 183–185
three-component mixture, resolution of 10-fold range of
decay times, 185
phase angle and modulation spectra, 189–191
phase modulation fluorescence equations, derivation of, 192–194
cross-correlation detection, 194
lifetime relationship to phase angle and modulation, 192–194
phase-modulation resolution of emission spectra, 197–199
phase-sensitive emission spectra, 194–197
examples of PSDF and phase suppression, 196–197
representative decays, 170–173
collisional quenching of chloride sensor, 171–172
exponential, 170–171
green fluorescent protein, one- and two-photon excitation, 171
intensity decay of NADH, 172
multi-exponential decays of staphylococcal nuclease and
melittin, 171
SPQ, collisional quenching, 171–172
scattered light, 172–173
simple frequency-domain instruments, 173–175
laser diode excitation, 174
LED excitation, 174–175
theory, 158–163
global analysis of data, 162–163
least-squares analysis of intensity decays, 161–162
Frequency-domain measurements
anisotropy decay, 417–418, 428–429, 588–589
excimer formation, 269–270
laser scanning microscopy, 750–751
protein distance distributions from, 485–487
time-domain lifetime measurements, 98–100
lifetime or decay time, meaning of, 99
phase modulation lifetimes, 99–100
Frequency doubling, 112
Frequency response, 160
Frequency synthesizers, 167
FRET. See Energy transfer
FR-GPI, 375–377
Front-face illumination, 55
FR-TM, 375–377
Fructose, 651
F-statistic, 133–134, 135, 136, 180
Fumarate, 278
Fura-2, 79, 648
Fusion proteins, 86
G
β-Galactosidase, 79
Gastrin, 590
Gated detection, 124–125
Gated-image intensifier, 747–748
Gating, 819
Gaussian distributions, 487
correlation time, 393
Gaussian lifetime distributions, 143
Gauss-Newton algorithms, 131
Gaviola, 163
GB1, 244–245
Generalized polarization, 218
Genetically engineered proteins
azurins, 538–539
for ribonuclease protein folding studies, 558–559
in sensors, 651–652
spectral properties, 554–557
barnase, 556–557
tyrosine proteins, 557
G-factor, 36, 362, 389
GFP, 86
anisotropy, 377–378
fluorescence-lifetime imaging microscopy, 747
GFP5, 747
GFP sensors, 654–655
Giant unilamellar vesicles (GUVs), 219, 465, 466
fluorescence correlation spectroscopy, 812
Gigahertz fluorometry, 175–178
Glan-Thompson polarizer, 50, 51
Global analysis
anisotropy decay
with multi-wavelength excitation, 429–430
frequency-domain lifetime measurements, 162–163, 182–183
time-domain lifetime measurements, 138, 144–145
Glucagon, 547, 548, 583
Glucose-galactose binding protein (GGBP), 652
Glucose sensors, 650–651
energy-transfer mechanisms, 634–635
Glutamate, 551
Glyceraldehyde phosphate, 555
Glyceraldehyde-3-phosphate dehydrogenase (GPD), 292, 600
Glycerol, 250, 359
Glycopeptides, 495–496
β-Glycosidase, 594
gly-trp-gly, 588
Gold
quenching, 313–315
molecular beacons, 723–724
surface plasmon-coupled emission, 867
Gold colloids, 845–846